ACTA CHROMATOGRAPHICA, NO. 13, 2003

NOVEL MOBILE PHASE FOR SEPARATION OF Cr6+ FROM Cr3+ AND ASSOCIATED HEAVY METAL

CATIONS BY HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY

A. Mohammad and Y. H. Sirwal Analytical Laboratory, Department of Applied Chemistry, Faculty of Engineering and Technology, Aligarh Muslim University, Aligarh-202002, India SUMMARY Eleven heavy metal cations have been chromatographed on silica gel high-performance thin-layer chromatography plates with pure organic, mixed organic, and mixed aqueous–organic mobile phases. Mobile phases such as methanol–dimethylamine, 8:2 (v/v), and methanol–dimethylamine –formic acid, 8:8:2 (v/v), were found most suitable for rapid separation and identification of mixtures of Cr6+ and Cr3+ and of Cr6+, Ni2+, and Co2+, respectively. The effect of impurities such as inorganic ions, phenols, and surfactants on the separation of Cr6+ and Cr3+ was examined. Mutual separation of Cr6+, Ni2+, and Co2+ was investigated at different sample solution pH. The limit of detection for Ni2+, Co2+, Cu2+, and Pb2+ on HPTLC plates was evaluated and semiquantitative determination of Cr6+ and Ni2+ by spot-area measurement was attempted. The proposed method was successfully used for identification of Cr6+, Cr3+, Ni2+, and Co2+ and for mutual separation of Cr6+ from Cr3+ and of Cr6+ from Ni2+ and Co2+ from a variety of industrial wastewater samples. INTRODUCTION Heavy metals have recently received considerable attention from analysts, because of their physical and environmental importance [1,2]. Metals such as Pb, Cd, Hg, Ni, Cu, Zn, As, and Cr6+ are toxic and harmful to human health. They can form stable complexes with bio-ligands containing oxygen, nitrogen, or sulphur atoms [3] which control several redox processes in living organisms. The substantial increase in the use of heavy metals over the past few decades has inevitably resulted in an increased flux of metallic substances in aquatic life. Industrial waste is the

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major source of different kinds of metal pollution in aqueous systems. The major sources of chromium in the aquatic environment are electroplating and metal-finishing industrial effluents, sewage and wastewater treatment plant discharge, and chromates from cooling water. Chromium occurs in several oxidation states (e.g. di-, tri-, penta-, and hexa-) but only Cr3+ and Cr6+ are biologically important. Chromium in the aquatic environment tends to speciate into Cr3+ and Cr6+, with the trivalent ion being oxidized to the hexavalent form or precipitating from solution. The different analytical techniques available for the detection, determination, and separation of chromium include normal-phase and reversed-phase thin-layer chromatography [4–6], ion chromatography [7,8], extraction chromatography [9], ion-exchange chromatography [10,11], reversed-phase high-performance liquid chromatography [12,13], micellar electrokinetic chromatography [14], precipitation flotation [15], solid-phase extraction [16], titrimetry [17,18], capillary electrophoresis [19,20], spectro-photometry [21,22], atomic-absorption spectroscopy [23–25], graphite furnace atomic-absorption spectroscopy [26,27], atomic emission spectro-scopy [28], neutron activation analysis [29,30], and hyphenated techniques such as ion-exchange chromatography–flame atomic absorption spectroscopy [31], ion-chromatography–thermal lens spectrometry [32,33], gas chroma-tography–neutron activation analysis [34], inductively coupled plasma mass spectroscopy [35,36], inductively coupled plasma mass spectroscopy –atomic emission spectroscopy [37,38], ion-exchange chromatography–flame atomic absorption spectroscopy [39], solid-phase extraction–flame atomic emission spectroscopy [40], liquid chromatography–inductively coupled plasma mass spectroscopy [41], high-performance liquid chroma-tography–inductively coupled plasma mass spectroscopy [42], and ion chromatography–inductively coupled plasma mass spectroscopy [43]. Of the different separation procedures, thin-layer chromatography (TLC) is probably the most versatile, because it can be used for the selective separation of metal cations on the micro and macro scales. The use of high-performance TLC plates has further enhanced the efficiency of this technique. An exhaustive survey of the literature published in the last thirty years [44] shows that much progress has been made in developing rapid and selective TLC methods for separation of toxic heavy metals (Cu, Ni, Co, Pb, Cd, Zn, Hg, Cr, Fe, and Al) from interfering elements, by use of a variety of acidic developers containing mineral or carboxylic acids as one of the components. Systematic examination of published data on the

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use of acidic mobile phases for analysis of metal cations shows that the number of applications of the acids decreases in the order:

HCl > HNO3 > H2SO4 > H3PO4 > CH3COOH > HCOOH > other carboxylic acids

That HCl is used most frequently is understandable, because it forms chloro complexes with almost all heavy metal cations. Perchloric acid has been used rarely. Use of formic acid as a mobile phase compo-nent in the TLC of metal cations has received little attention [45–49] despite several favourable properties: (i) it does not result in oxidation of cations during analysis; (ii) formic acid-containing mobile phases are less affected by the properties of silica gel than those containing other acids [50]; (iii) it enables excellent resolution of aflatoxins [50] and metal cations [45–49]; and (iv) it is sufficiently acidic (Ka (H2O) at 25°C = 1.77 × 10–4) to prevent hydrolysis of salts. All studies with formic acid-containing mobile phases have been performed using conventional, laboratory made TLC plates. It was there-fore decided to use the analytical potential of formic acid as mobile phase and precoated HPTLC silica plates as stationary phase for analysis of heavy metal cations. As a result several analytically important separations of heavy metals were realized. Separation of the different valence states of chromium is industrially important because Cr3+ is converted to Cr6+ in alkaline peroxide media. EXPERIMENTAL Chemicals and Reagents

Dimethylamine was from S.D. Fine Chemicals (India), dimethyl-aniline, inorganic salts, amines, and phenols were from CDH (India), o-aminophenol was from Loba Chemie (India), formic acid was from Merck (India), and methanol and acetone were from Qualigens (India). All reagents were analytical reagent grade. Test Solutions

Test solutions, prepared in double-distilled water, contained 1.0% aqueous solutions of the nitrates of Cd2+, Pb2+, Tl+, Bi3+, Al3+, and Ag+, the chlorides of Ni2+, Co2+, Hg2+, and Cr3+, the sulphate of VO2+, and the

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potassium salt of Cr6+. The solutions of the nitrates of lead, silver, and bismuth, and the chloride of mercury contained small quantities of the corresponding acid to limit the extent of hydrolysis. Detection Reagents

Yellow ammonium sulphide reagent was used for detection of Cd2+, Ag+, Pb2+, Tl+, Bi3+, and Hg2+, a 1% aqueous solution of potassium ferrocyanide for detection of VO2+; dimethylglyoxime (0.2% in ammonia) for detection of Ni2+ and Co2+, a saturated alcoholic solution of AgNO3 for detection of Cr6+, and a 1% methanolic solution of alizarin red for detection of Cr3+. Chromatography

Chromatography was performed on silica gel 60 F254 HPTLC plates (Merck, Darmstadt, Germany). Test solutions (5 µL) were applied approximately 2.0 cm above the lower edge of the plates by means of a micropipette. The spots were dried and the plates were developed in glass jars by the one-dimensional ascending technique. The mobile phases investigated are listed in Table I. Before development of the plates the glass jars containing the mobile phases were covered with a lid for approximately 20 min to enable presaturation of the glass jars with mobile phase vapour. The mobile phase migration distance was always 10 cm from the starting line. After development the plates were dried and the cations were visualized as coloured spots by spraying with the appropriate detection reagents. The cations were identified on the basis of their RF values, calculated from RL (RF of the leading front) and RT (RF of trailing front) for each spot. Separations

Test solutions (10 µL) containing the metal ions to be separated were spotted on the HPTLC plates and chromatography was performed with a variety of mobile phases. The resolved spots for these metal cations were observed on the plates after spraying with chromogenic reagents and the RF values of the separated metal ions were determined. Interference

To investigate the effect of interference of inorganic ions, phenols, and surfactants on the RF values (mobility) of Cr6+ and Cr3+ a solution of the impurity (10 µL) was spotted with each metal ion, as a mixture, on the

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HPTLC plate and chromatography was performed as described above. The spots were detected and the RF values of separated metal ions were determined. Table I

The mobile phases investigated

Symbol Composition M1 Methanol (MeOH) M2 Dimethylamine (DMA) M3 Formic acid (FA) M4 Acetone M5 Dimethylaniline (DMAL) M6 ortho-Aminophenol (o-APH) M7 Methanol–dimethylamine, 8:2 M8 Methanol–formic acid, 8:2 M9 Water–dimethylamine, 8:2 M10 Water–formic acid, 8:2 M11 Dimethylamine–methanol–formic acid, 2:8:2 M12 Dimethylamine–methanol–formic acid, 4:8:2 M13 Dimethylamine–methanol–formic acid, 8:8:2 M14 Dimethylamine–methanol–formic acid, 10:8:2 M15 Dimethylamine–acetone–formic acid, 2:8:2 M16 Dimethylamine–acetone–formic acid, 4:8:2 M17 Dimethylamine–acetone–formic acid, 8:8:2 M18 Dimethylamine–acetone–formic acid, 10:8:2 M19 Dimethylamine–water–formic acid, 2:8:2 M20 Dimethylamine–water–formic acid, 4:8:2 M21 Dimethylamine–water–formic acid, 8:8:2 M22 Dimethylamine–water–formic acid, 10:8:2 M23 Dimethylaniline–methanol–formic acid, 2:8:2 M24 Dimethylaniline–methanol–formic acid, 4:8:2 M25 Dimethylaniline–methanol–formic acid, 8:8:2 M26 Dimethylaniline–methanol–formic acid, 10:8:2 M27 o-Aminophenol–methanol–formic acid, 2:8:2 M28 o-Aminophenol–methanol–formic acid, 4:8:2 M29 o-Aminophenol–methanol–formic acid, 8:8:2 M30 o-Aminophenol–methanol–formic acid, 10:8:2

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Effect of pH

The effect of pH on the mutual separation of Cr6+, Ni2+, and Co2+ was investigated by adding required amount of acid to a mixed solution of the three metals and analysing the sample by HPTLC as described above. Limit of Detection

Detection limits for Ni2+, Co2+, Cu2+, and Pb2+ were determined by spotting different amounts of solutions of the cations on the HPTLC plates, developing the plates with mobile phase M7 (Table I), and detecting the spots as described above. The method was repeated with successive reduction of the amounts of the cations until spots could no longer be detected. The minimum amount of cation that could be detected was taken as the limit of detection. Semi-Quantitative Determination by Spot-Area Measurement

For semi-quantitative determination by spot-area measurement standard solutions (0.5–2.0%, 10 µL) of Ni2+ and Cr6+ were spotted on the HPTLC plates and the plates were developed with mobile phases M7 and M13 (Table I). After detection the spots were copied on to tracing paper from the plates and then the area of each spot was calculated. Semi-Quantitative Determination by Visual Comparison

Standard solutions of potassium dichromate of different concentra-tions (0.5–2.0%, 10 µL), and an industrial wastewater sample (10 µL), were spotted on HPTLC plates. After completion of the chromatography the colour intensity and RF values of spots from the industrial wastewater sample were matched with the coloured spots obtained from standard reference solutions of potassium dichromate. The amount of chromium present in the industrial sample was determined from the colour intensity of its spot on the plate after visual comparison with the colour intensities of the spots obtained from standard solutions. Chromatography of Spiked Wastewater Samples

Three spiked samples of industrial wastewater were prepared and chromatographed. Wastewater sample 1 containing Cr6+ was spiked with an aqueous solution of Cr3+ (1%) in a 1:1 (v/v) ratio. The resulting spiked sample (ca. 10 µL) was analysed by HPTLC with M7 as mobile phase and the RF values of the resolved spots of Cr6+ and Cr3+ were determined.

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Industrial wastewater samples containing Ni2+ (samples 2 and 3) were spiked with aqueous solutions of Cr6+ (1%) and Cr3+ (1%) in a 1:1:1 (v/v) ratio and 10 µL of the resulting solution was analysed by HPTLC with M13 as mobile phase. The RF values of resolved spots of Ni2+, Cr6+, and Cr3+ were determined. RESULTS AND DISCUSSION The results from this study have been summarized in Tables II–VIII and Figs 1–3. The mobilities of metal cations chromatographed with pure single-component organic mobile phases (M1–M6), two-component mixed organic mobile phases (M7 and M8) and aqueous–organic mobile phases (M9 and M10) have been summarized in Table II. It is clear from this table that, except for Cr6+, only formic acid-containing mobile phases (M3, M8, and M10) induce migration of the metal cations; Cr6+ is more mobile in some mobile phases (M1–M4, M7, and M9) which contain no formic acid. The use of pure acetone as mobile phase (M4) resulted in the Table II

Mobility (as RF value) of heavy metal ions on silica gel HPTLC plates developed with single-component (M1–M6), two-component organic (M7, M8) and two-component aqueous–organic (M9, M10) mobile phases

Mobile phase Metal ion M1 M2 M3 M4 M5 M6 M7 M8 M9 M10

VO2+ 0.00 0.00 0.00 0.00 0.00 0.08 0.00 0.00 0.00 0.00

Cr6+ 0.34 0.86a 0.72 0.00, 0.97b 0.01 0.00 0.86 0.63 0.85 0.78

Ag+ 0.00 0.00 0.15 0.00 0.03 0.02 0.00 0.15 0.00 0.09 Hg2+ 0.45Tc 0.00 0.85 0.00 0.10 0.02 0.30 0.82 0.04 0.94 Pb2+ 0.00 0.00 0.68 0.00 0.00 0.00 0.00 0.49 0.10 0.93 Cd2+ 0.08 0.00 0.78 0.00 0.05 0.05 0.02 0.72 0.00 0.94 Tl+ 0.10 0.00 0.82 0.00 0.00 0.00 0.05 0.76 0.00 0.78 Ni2+ 0.05 0.00 0.87 0.00 0.00 0.04 0.02 0.81 0.00 0.84 Co2+ 0.04 0.06 0.75 0.00 0.00 0.03 0.04 0.68 0.00 0.88 Bi3+ 0.09 0.00 0.16 0.09 0.02 0.40Tc 0.06 0.16 0.04 0.23 Cr3+ 0.05 0.05 0.70 0.05 0.05 0.05 0.05 0.60 0.05 0.75

a Detection clarity is poor b Double spot c Tailed spot (RL – RT > 0.3)

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formation of double spots for Cr6+. The peculiar behaviour of formic acid as tailing reducer and mobility activator results in new opportunities for separation of metal cations. With this in mind we added a third component – methanol, acetone, or water – to the mixtures of amines and formic acid and the resulting three-component mixtures (M11–M26) were investigated as mobile phases for TLC of metal cations. Amines were selected on the basis of our past experience [51] that they furnish highly compact and well-resolved spots of metal cations on silica layers. The results obtained by use of mixed aqueous–organic mobile phases (M11–M22) containing different concentrations of DMA and fixed concentrations of formic acid and organic modifier (M11–M18) or water (M19–M22) are tabulated in Table III. On the basis of these results, the metal Table III

Migration behaviour of heavy metal cations on precoated silica gel HPTLC plates with dimethylamine, formic acid, and/or acetone, methanol, water in different volume ratios as mobile phases

Mobile phase Metal ion M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 M21 M22

VO2+ 0.00 0.00 0.00 0.00 0.00 0.00 0.03 0.00 0.05 0.02 0.00 0.00 Cr6+ 0.62 0.65 0.91 0.92 0.55 0.63 0.68 0.90 0.53 0.53 0.59 0.89 Ag+ 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.13 0.11 0.00 Hg2+ 0.82 0.72 0.62 0.52 0.88 0.86 0.79 0.76 0.95 0.59 0.17 0.10 Pb2+ 0.63 0.52 0.28 0.15 0.84 0.81 0.77 0.00 0.86 0.57 0.02 0.00 Cd2+ 0.89 0.66 0.42 0.29 0.84 0.80 0.78 0.76 0.93 0.84 0.26 0.09 Tl+ 0.86 0.78 0.13 0.10 0.78 0.61 0.09 0.04 0.78 0.64 0.35 0.02 Ni2+ 0.87 0.68 0.44 0.25 0.77 0.68 0.56 0.42 0.93 0.84 0.23 0.00 Co2+ 0.81 0.66 0.05 0.00 0.73 0.67 0.42 0.00 0.89 0.81 0.35 0.00 Bi3+ 0.15 0.08 0.00 0.00 0.14 0.13 0.05 0.00 0.15 0.11 0.06 0.03 Cr3+ 0.60 0.68 0.90 0.90 0.53 0.75 0.70 0.90 0.50 0.55 0.60 0.86

cations can be grouped into three categories. The metal cations VO2+, Ag+, and Bi3+ were strongly retained by the stationary phase and remained near the point of application, irrespective of the concentration of dimethylamine and the nature of the organic modifier (methanol or acetone). The mobility of the metal cations Cr3+ and Cr6+ increased with increasing concentration of dimethylamine in the mobile phase, irrespective of whether the mobile phase contained methanol (M11–M14), acetone (M15–M18), or water (M19–M22) in combination with formic acid and

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dimethylamine. The RF of the metal cations Hg2+, Pb2+, Cd2+, Tl+, Ni2+ Co2+, and Bi3+ decreased with increasing concentration of dimethylamine in the mobile phases (M11–M22). Thus, these mobile phases facilitate selective separation of several metal cations by virtue of the variable mobility of the ions. For example, Cr3+ and Cr6+ can be selectively separated from all other metal cations by use of M13, M14, M18, M21, and M22, because their mobility in these mobile phases is higher than that of the other metal cations. Similarly, M13 and M18 can be used to achieve the analytically important separation of Ni2+, Co2+, and Cr6+ from their mixtures. This separation could not be achieved with pure organic (M1–M6), two-component mixed organic (M7, M8), or mixed aqueous–organic (M9, M10) mobile phases. To examine the effect of the nature of the amino compounds on the mobility of metal cations, dimethylamine was replaced by dimethylaniline (M23–M26) and ortho-aminophenol (M27–M30) in mobile phases M11–M14 while maintaining the volume ratio of methanol and formic acid the same. The RF values of the metal cations were determined after use of the resulting mobile phases (M23–M30). The results obtained are encapsulated in Table IV. It is clear from this table that the nature of amino compound has a pronounced effect on the RF (or mobility) of the metal cations. Table IV

RF values of metal cations on silica gel HPTLC plates developed with mixed three-component mobile phases consisting of methanol, formic acid, and dimethylaniline or o-aminophenol in different volume ratios

Mobile phase Metal ion M23 M24 M25 M26 M27 M28 M29 M30

VO2+ 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Cr6+ 0.60 0.56 0.07 0.05 0.41 0.81 0.83 0.86 Ag+ 0.15 0.15 0.12 0.05 0.15 0.12 0.04 0.02 Hg2+ 0.72 0.66 0.56 0.42 0.80 0.64 0.52 0.40 Pb2+ 0.61 0.27 0.15 0.06 0.25 0.25 0.30 0.32 Cd2+ 0.48 0.39 0.28 0.15 0.55 0.65 0.75 0.85 Tl+ 0.85 0.80 0.62 0.41 0.70 0.60 0.48 0.32 Ni2+ 0.72 0.64 0.52 0.16 0.64 0.87 0.90 0.92 Co2+ 0.70 0.52 0.42 0.30 0.60 0.64 0.70 0.76 Bi3+ 0.16 0.10 0.09 0.07 0.15 0.17 0.17 0.18 Cr3+ 0.60 0.55 0.10 0.05 0.40 0.60 0.80 0.82

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VO2+, Ag+, and Bi3+ are poorly mobile in all the mobile phases. Several changes in the RF values of the cations resulted from substitution of dimethylamine with dimethylaniline – the RF of Cr6+ dropped from 0.91 (M13) and 0.92 (M14) to 0.07 (M25) and 0.05 (M26), respectively; the RF of Pb2+ decreased from 0.52 (M12) to 0.27 (M24); the RF of Cd2+ decreased from 0.89 (M11), 0.66 (M12), 0.42 (M13), and 0.29 (M14) to 0.48 (M23), 0.39 (M24), 0.28 (M25), and 0.15 (M26), respectively; the RF of Tl+ increased from 0.13 (M13) and 0.10 (M14) to 0.62 (M25) and 0.41 (M26), respectively; and the RF of Cr3+ decreased from 0.90 (M13 and M14) to 0.10 (M25) and 0.05 (M26), respectively. Similarly, substitution of dimethylaniline in M11–M14 by o-amino-phenol caused a decrease in the RF value of Pb2+ from 0.63 (M11) and 0.52 (M12) to 0.25 (M27, M28) whereas the RF of Cd2+ increased from 0.29 (M14) to 0.85 (M30). The RF values of Ni2+ and Co2+ increased from 0.00 (M14) to 0.92 and 0.76 (M30), respectively. A decrease in the RF of Cr3+ from 0.60 (M11) to 0.40 (M27) was also observed. It is clear from these observations that amine–methanol–formic acid mobile phases have enormous analytical potential for achieving selective separations of heavy metal cations from their multi-component mixtures, because the nature of the added amine has a profound influence on the mobility of cations. Some separations of metal cations achieved experimentally using different mobile phases have been encapsulated in Table V. Tables VI and VII summarize the effects of a variety of inorganic ions, surfactants, and phenolic impurities on the separation of mixtures of Cr6+ and Cr3+. It is evident from Table VI that inorganic ions result in a slight change in the mobility of Cr6+ without influencing the mobility of Cr3+. Thus, separation is always possible. Among the heavy metal ions, Hg2+ and Al3+ affected the mobility of Cr6+, resulting in the formation of tailed spot. The presence of MoO4

2– significantly reduced the RF value of Cr6+ – from 0.85 to 0.71. Cr6+ also formed a tailed spot in the presence of Br–. The results presented in Table VII clearly show that the effect of surfactants on the mobility of chromium ions is similar to that of inorganic ions. The mobility of Cr3+ was unaffected whereas that of Cr6+ changed slightly. The compact Cr6+ spot became elongated in the presence of surfactant, irrespective of the nature of the latter (anionic, cationic, or non-ionic). Despite this, separation of Cr6+ from Cr3+ was always possible. Phenolic impurities also affected the mobility of Cr6+ slightly whereas the mobility of Cr3+ remained unaffected. Separation of Cr6+ from Cr3+ was

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Table V

Experimentally achieved separations of heavy metal cations on silica gel HPTLC plates developed with different mobile phases

Mobile phase Separations

Formic acid Hg2+ or Ni2+ (0.86)/Cd2+ or Co2+(0.75)/Cr6+ or Cr3+ (0.71)/Pb2+ (0.67)–VO2+ (0.00)/Bi3+ (0.15)

MeOH–DMA, 8:2 Cr6+(0.85)–Cr3+ (0.05) Cr6+ (0.85)–VO2+, Ag+, or Pb2+ (0.00)/Cd2+

or Ni2+ (0.02)/Co2+, Tl+ or Bi3+ (0.05) MeOH–formic acid,

8:2 Hg2+ or Ni2+ (0.81)/Cd2+ (0.71)/Tl (0.75)/Cr6+

or Cr3+ (0.76)–Bi3+ or Ag+ (0.15)/VO2+ (0.00) Water–formic acid,

8:2 Hg2+, Pb2+, or Cd2+(0.93)/Ni2+or Co2+ (0.86)/Cr6+

or Cr3+ (0.76)–Bi3+ (0.21)/Ag+ (0.10)/VO2+(0.00)

DMA–MeOH–formic acid, 8:8:2

Cr6+ (0.90)–Ni2+ (0.45)–Co2+ (0.00) Cr6+ or Cr3+ (0.90)–Hg2+ (0.60)/Cd2+–VO2+, Ag+,

or Bi3+ (0.00) DMA–acetone–

formic acid, 2:8:2 Hg2+ (0.88)/Pb2+ or Cd2+ (0.85)/Tl+ or Ni2+ (0.78)–Cr6+

or Cr3+ (0.54)–Bi3+ (0.15)/VO2+ or Ag+ (0.00)

DMA–acetone–formic acid, 10:8:2

Cr6+ (0.90)–Ni2+ (0.40)–Co2+ (0.00) Cr6+ or Cr3+ (0.90)/Hg2+ or Tl+ (0.75)–VO2+, Ag+, Pb2+,

or Bi3+ (0.00) DMA–water–formic

acid, 10:8:2 Cr6+ or Cr3+ (0.87)–Hg2+ or Cd2+ (0.10)/VO2+, Ag+,

Pb2+, Ni2+, or Co2+ (0.00) o-APH–MeOH–

formic acid, 10:8:2 Ni2+ (0.92)/Cr6+ or Cd2+ (0.85)–Hg2+ (0.39)/Pb2+

or Tl+ (0.33)–Bi3+ (0.16)/VO2+ (0.05) or Ag+ (0.02) possible in the presence of all the phenols except p-aminophenol, which caused significant tailing of Cr6+ and hampered its separation from Cr3+. The effect of pH on the mutual separation of Ni2+, Co2+, and Cr6+ is shown in Fig. 1. It is clear from this figure that the best separation of a mixture of Ni2+, Co2+, and Cr6+ was achieved in the pH range 2.5–3.5. At pH 1.5 the mobility of Ni2+ was reduced such that it could not be separated from Co2+, whereas it is well separated from Cr6+. Conversely, at pH 6.0 the RF of Ni2+ was increased to such an extent that its separation from Cr6+ was not possible but its separation from Co2+ is always possible. At pH 8.0 the mobility of Ni2+ was further increased and the ion co-migrated with Cr6+. The lowest detectable amounts (µg) and dilution limits of metal ions achieved on HPTLC plates developed with mobile phase M7 were,

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Table VI

Effect of inorganic impurities on the separation of Cr6+ and Cr3+ from their mixture on silica gel HPTLC plates developed with mobile phase M7

Separation (RF value) Impurities

Cr6+ Cr3+ Ni2+ 0.83 0.05 Co2+ 0.83 0.05 Cd2+ 0.78 0.05 Zn2+ 0.79 0.05 Ag+ 0.84 0.05 Pb2+ 0.84 0.05 Tl+ 0.88 0.05 Bi3+ 0.78 0.05 Hg2+ 0.80Ta 0.05 Al3+ 0.80Ta 0.05 NaNO2 0.75 0.05 NaNO3 0.79 0.05 NaH2PO4 0.79 0.05 KI 0.81 0.05 KIO3 0.90 0.05 KBr 0.67Ta 0.05 K3[Fe(CN)6] 0.81 0.05 K4[Fe(CN)6] 0.86 0.05 NH4SCN 0.81 0.05 (NH4)2 MoO4 0.71 0.05 Without impurity 0.85 0.05

a Tailed spot (RL – RT > 0.30) respectively, 0.028 and 1:3.5 × 105 for Ni2+, 0.11 and 1:9 × 104 for Co2+, 2.09 and 1:4.784 × 103 for Cu2+, and 0.08 and 1: 1.25 × 105 for Pb2+. It is clear from these data that the proposed method is a highly sensitive means of detection of the cations. In addition to qualitative analysis, quantitative evaluation of the metal ions is often required to ascertain the levels of toxic metals in environ-mental samples. An approximate but simple method of quantitation is based on measurement of spot size by drawing the outline of the spot on a piece of tracing paper. Semi-quantitative determination of metal ions by measurement of spot area was therefore attempted. The linear relationship

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Table VII

Effect of surfactants and phenolic impurities on the separation of Cr6+ and Cr3+ from their mixture on silica gel HPTLC plates developed with mobile phase M7

Retention (RF value) Impurity

Cr6+ Cr3+ Sodium dodecyl sulphate (SDS) 0.67Ta 0.05 N-Cetyl-N,N,N-trimethylammonium bromide (CTAB) 0.64Ta 0.05 Polyoxyethylene dodecyl ether (Brij-35) 0.75Ta 0.05 Polyoxyethylene (20) cetyl ether (Brij-58) 0.67Ta 0.05 Polyoxyethylene (20) stearyl ether (Brij-78) 0.70Ta 0.05 Polyoxyethylene (20) oleyl ether (Brij-98) 0.67Ta 0.05 Polyoxyethylene (20) sorbitan monolaurate 0.75 0.05 Polyoxyethylene (4) sorbitan monopalmitate 0.67Ta 0.05 Sorbitan monostearate (Span-60) 0.69Ta 0.05 Orcinol 0.80 0.05 Resorcinol 0.76 0.05 Pyrocatechol 0.80 0.05 Phloroglucinol 0.83 0.05 Pyrogallol 0.81 0.05 o-Aminophenol 0.80 0.05 m-Aminophenol 0.86 0.05 p-Aminophenol 0.50Ta 0.05 Without impurity 0.85 0.05

a Tailed spot (RL – RT > 0.30) obtained when the amount of the sample spotted was plotted against the area of the spot (Figs 2 and 3) followed the empirical equation ξ = k × m, where ξ is the area of the spot, m the amount of sample, and k a constant. Linearity is maintained up to 200 µg/spot of Ni2+ and Cr6+. At higher con-centrations a negative deviation from the linear law was observed for both metals. The standard curve constructed for semi-quantitative determination of Cr6+ (Fig. 2) was used to determine the amount of chromium present in a sample of industrial wastewater. Accuracy and precision were below ±15%. Semi-quantitative determination by visual comparison was used to estimate the amount of Cr6+ present in industrial wastewater. The industrial wastewater samples analysed (chrome wastewater) were found to have a chromium content in the range 5–75 µg L–1.

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Fig. 1

Effect of pH on the separation of Cr6+, Ni2+, and Co2+ by use of in M13 as mobile phase on silica gel HPTLC plates

Fig. 2

Calibration plots for semiquantitative determination of Cr6+ and Ni2+ on silica gel HPTLC plates developed with mobile phase M7

The proposed method was successfully applied for identification and separation of heavy metal ions in spiked industrial wastewater samples. The results presented in Table VIII clearly demonstrate the applicability of the method for identification of Cr6+, Cr3+, Ni2+, and Co2+in a variety of industrial wastewater samples.

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Fig. 3

Calibration plots for semiquantitative determination of Ni2+ and Cr6+on silica gel HPTLC plates developed with mobile phase M13

Table VIII

Application of proposed method (HPTLC on silica gel with mobile phases M7 and M13) for separation and identification of heavy metals from industrial wastewater

Mobile phase Industrial waste Separation (RF) M7 Sample 1 Cr6+ (0.86)–Ni2+ (0.45)–Co2+ (0.00) M13 Sample 2 Cr6+ (0.87)–Ni2+ (0.41)–Co2+ (0.03) M13 Sample 3 Cr6+ (0.87)–Ni2+ (0.43)–Co2+ (0.00)

ACKNOWLEDGMENT The authors are thankful to the Chairman, Department of Applied Chemistry, for providing research facilities, and the All India Council for Technical Education, New Delhi (India) is thanked for providing financial assistance to perform this work. REFERENCES

[1] H. Siegel, Metal Ions in Biological Systems, Vols. I and II, Marcel Dekker, New York, USA, 1986

[2] V. Venngopal and T. Luckey, Metal Toxicity of Metals and Metalloids, Plenum, New York, USA, 1987

- 131 -

[3] M. Bukowska – Strzyżewska, W. Maniukewicz, G. Bazylak, and J. Masłowska, J. Crystallogr. Spectrosc. Res., 21, 157 (1991)

[4] G. Schubert, V. Alan, J. Zirko – Babic, and S. Turina, J. Planar Chromatogr., 11, 460 (1998)

[5] S. Przeszlakowski and M. Maliszewska, Chem. Anal., 37, 547 (1992) [6] S.D. Sharma and S.C. Sharma, J. Chromatogr., A 841, 263 (1999) [7] M. Mizobuchi, Y. Horie, and K. Saito, Nara-ken Eisei Kenkyusho

Nenpo, 26, 56 (1991) (Published 1992); Chem. Abstr., 118, 20248q (1993)

[8] Y. Zhou, G. Shao, and S. Mu, Huanjing Huaxue, 15, 446 (1996); Chem Abstr., 126, 36660z (1997)

[9] R.O. Crubellati and A.G. Ledesma, Analyst, 118, 529 (1993) [10] S.A. Nabi, A. Gupta, and A. Sikarwar, Ann. Chim., 89, 419 (1999) [11] M. Ghannadi – Marageh, S.W. Husain, A.R. Khanchi,

and S.J. Ahmady, Sep. Sci. Technol., 34, 219 (1999) [12] M.L. Marina, P. Andres, and J.C. Diez – Masa, Chromatographia,

35, 621 (1993) [13] Y. Tomita, Y. Aoyama, and E. Aoyama, Ibaraki Kogyo Koto

Senmon Gakko Kenkyu Iho, 36, 65 (2001); Chem. Abstr., 135, 50686e (2001)

[14] E.F. Hilder, M. Macka, and P.R. Haddad, Analyst, 123, 2865 (1998) [15] K.S. Lee, H.S. Choi, and Y.S. Kim, Anal. Sci. Technol., 3, 419

(1990) [16] V. Ostruba, J. Pivnicka, and V. Kanicky, Collect. Czech. Chem.

Commun., 65, 1865 (2000) [17] L. Zheng and S. Wang, Yejin Fenxi, 16, 49 (1996); Chem. Abstr.,

126, 165878a (1997) [18] K.K. Paliwal, D.K. Gorji, S. Kumar, N. Naulakha, A.K. Goswami,

and D.N. Purohit, Asian J. Chem., 13, 299 (2001) [19] M.C. Mehra and C.A. Lucy, Orient. J. Chem., 12, 231 (1996) [20] Z. Chen, R. Naidu, and A. Subramanian, J. Chromatogr. A,

927, 219 (2001) [21] W. Shi and T. Hu, Huaxue Fence, 34, 447 (1998); Chem. Abstr.,

130, 85763b (1999) [22] Z.G. Xu, X.H. Wen, and Q.Z. Wu, Chin. Chem. Lett., 12, 635

(2001) [23] Z. Shen, Y. Li, and Y. Li, Guangdong Weiliang Yuansu Kexue,

5, 69 (1998); Chem. Abstr., 130, 147940t (1999)

- 132 -

[24] D. Qin, Z. Lin, and J. Wang, Huaxue Fenxi Jiliang, 9, 17 (2002); Chem. Abstr., 135, 24284c (2001)

[25] A. Gaspar, C. Songor, and J. Posta, Fresenius J. Anal. Chem., 363, 480 (1999)

[26] S.T. Sauerhoff, Z.A. Grosser, and G.R. Carnrick, At. Spectrosc., 17, 225 (1996)

[27] T.W. Lin and S.D. Huang, Anal. Chem., 73, 4319 (2001) [28] E.S. Zolotovitskaya, L.V. Glushkova, Z.V. Shititelman,

and A.B. Blank, Khim. Tekhnol. Vody, 15, 255 (1993) [29] T.M. Bahrainwala and Z.R. Tural, J. Radioanal. Nucl. Chem.,

214, 199 (1996) [30] H. Rausch and T. Braun, Fullerene Sci. Technol., 5, 407 (1997) [31] B. Demirata, I. Tar, H. Filik, and H. Afsar, Fresenius J. Anal.

Chem., 356, 375 (1996) [32] M. Sikovec, M. Franko, and M. Novic, AIP Conf. Proc.,

463, 682 (1999) [33] M. Sikovec, M. Franko, M. Novic, and M. Veber, J. Chromatogr. A,

920, 119 (2001) [34] G.S. Sattarov and A.A. Kist, Czech. J. Phys., 49, 303 (1999) [35] T. Etoh, M. Yamada, and M. Matsubara, Kankyo Kagaku,

3, 398 (1993); Chem. Abstr., 119, 102860y (1993) [36] J.C. Yu, X.-J. Wu, and Z. Chen., Anal. Chim. Acta, 436, 59 (2001) [37] J. Guan, Y. Shi, B. Gao, and Y. Liu, Yuanzineng Kexue Jishu,

25, 8 (1991); Chem. Abstr., 118, 15539m (1993) [38] H. Chen, Huaxue Shijie, 41, 488 (2000); Chem. Abstr., 134, 36428v

(2001) [39] V. Stresko, J. Polakovicova, and A. Celkova, Chem. Pap.,

55, 100 (2001) [40] V. Ostruba, J. Pivnicka, and V. Kanicky, Proc. – Semin. At.

Spectrochem., 14, 247 (1998) [41] Y.-L. Chang and S.J. Jiang, J. Anal. At. Spectrom., 16, 858 (2001) [42] Y. Martinez – Bravo, A.F. Roig – Navaro, F.J. Lopez,

and F. Hernandez, J. Chromatogr., A 926, 256 (2001) [43] H. Guerleyuek and D. Wallschlaeger, J. Anal. At. Spectrom.,

16, 926 (2001) [44] A. Mohammad, M. Ajmal, S. Anwar, and E. Iraqi, J. Planar

Chromatogr., 9, 318 (1996) [45] N. Fatima and A. Mohammad, Sep. Sci. Technol., 19, 429 (1994)

- 133 -

[46] M. Ajmal, A. Mohammad, and N. Fatima, Microchem. J., 37, 314 (1988)

[47] A. Mohammad and N. Fatima, Chromatographia, 22, 109 (1986) [48] A. Mohammad and M.A.M. Khan, J. Planar Chromatogr.,

8, 134 (1995) [49] A. Mohammad, M. Ajmal, and S. Anwar, Acta Chromatogr.,

9, 113 (1999) [50] I. Balzer, C. Bogdanic, and C. Pepeljnjak, J. Assoc. Anal. Chem.,

61, 584 (1978) [51] M. Ajmal, A. Mohammad, N. Fatima, and J. Ahmad, J. Planar

Chromatogr., 1, 329 (1988)

- 134 -